Differential interactions of missing in metastasis and insulin receptor tyrosine kinase substrate with RAB proteins in the endocytosis of CXCR4

Abstract

Edited by Phyllis I. HansonMissing in metastasis (MIM), an inverse Bin-Amphiphysin-Rvs (I-BAR) domain protein, promotes endocytosis of C-X-C chemokine receptor 4 (CXCR4) in mammalian cells. In response to the CXCR4 ligand stromal cell-derived factor 1 (SDF-1 or CXCL12), MIM associates with RAS-related GTP-binding protein 7 (RAB7) 30 min after stimulation. However, RAB7's role in MIM function remains undefined. Here we show that RNAimediated suppression of RAB7 expression in human HeLa cells has little effect on the binding of MIM to RAB5 and on the recruitment of CXCR4 to early endosomes but effectively abolishes MIM-mediated CXCR4 degradation, chemotactic response, and sorting into late endosomes and lysosomes. To determine whether I-BAR domain proteins interact with RAB7, we examined cells expressing insulin receptor tyrosine kinase substrate (IRTKS), an I-BAR domain protein bearing an Src homology 3 (SH3) domain. We observed that both MIM and IRTKS interact with RAB5 at an early response to SDF-1 and that IRTKS binds poorly to RAB7 but strongly to RAB11 at a later time point. Moreover, IRTKS overexpression reduced CXCR4 internalization and enhanced the chemotactic response to SDF-1. Interestingly, deletion of the SH3 domain in IRTKS abolished the IRTKS-RAB11 interaction and promoted CXCR4 degradation. Furthermore, the SH3 domain was required for selective targeting of MIM-IRTKS fusion proteins by both RAB7 and RAB11. Hence, to the best of our knowledge, our results provide first evidence that the SH3 domain is critical in the regulation of specific endocytic pathways by I-BAR domain proteins.

IRSp53 controls plasma membrane shape and polarized transport at the nascent lumen in epithelial tubules

“…Our proteomic studies using IRSp53–BirA approaches that can detect transient interactions 64 , however, failed to identify any of these proteins {deposited at PASS01464 , for details see Methods}, which would argue against this mode of action. Finally, it is of note that also other I-BAR domain family members, IRTKS and MIM, have been shown to interact with RAB GTPases and to be involved in endocytosis 65 . Thus, the involvement in membrane trafficking appears to be a more general emerging function of the I-BAR family proteins.…”